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W C Kenney

Publications and source records attributed to W C Kenney.

At least 19 recordsLinked to original sources
PubMed2001

Factors affecting short-term and long-term stabilities of proteins.

Proteins are marginally stable and, hence, are readily denatured by various stresses encountered in solution, or in the frozen or dried states. Various additives are known to minimize damage and enhance the stability of proteins. This review discusses the current knowledge of the mechanisms by which these additives stabilize proteins against acute stresses, and also the various factors to be considered for long-term storage of proteins in solution.

Drug Stability↗
PubMed1998

Human keratinocyte growth factor recombinantly expressed in Chinese hamster ovary cells: isolation of isoforms and characterization of post-translational modifications.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.

Amidohydrolases↗
PubMed1996

Characterization of keratinocyte growth factor binding to heparin and dextran sulfate.

Binding of keratinocyte growth factor (KGF) with heparin (molecular weight of 5000) and dextran sulfate (molecular weight of 8000) was studied using an online monitoring of size-exclusion chromatography with light scattering, refractive index, and uv absorbance detectors. This technique allows the determination of the molecular weight of KGF eluting as complexes with the above polymers. When mixtures of KGF with heparin were injected into the column, two peaks of heparin/KGF complexes were observed. The first peak corresponded to, on average, 3.4 KGF per complex and the second peak to an average of about 2 KGF per complex. These results suggest that the heparin/ KGF complex is heterogeneous, consisting of 1,2,3, and 4 KGF molecules per complex. To calculate the number of heparin molecules in these complexes, the rate of disappearance of free KGF was determined as heparin was added. The average number of KGF bound to 1 mol of heparin was calculated to be about 2 mol, suggesting that only one heparin molecule is present in these complexes. The heparin binding of two KGF mutants, i.e., (C1, 15S)KGF (with substitutions of serine for cysteines 1 and 15) and d28KGF (lacking 28 N-terminal amino acid residues), was essentially identical to that of the native sequence KGF. A similar experiment was carried out for KGF binding to dextran sulfate. The molecular weight of the complex corresponded to 2 to 2.6 KGF molecules per complex. The rate of disappearance of free KGF as the dextran sulfate added showed 2-3 mol of KGF bound to 1 mol of dextran sulfate, consistent with the idea that the complex contains only 1 dextran sulfate molecule.

Binding Sites↗
PubMed1995

Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli.

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Amino Acid Sequence↗
PubMed1995

Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds.

Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.

Adult↗
PubMed1995

Pharmacokinetic, pharmacodynamic, and pharmacotoxic profiles of recombinant-methionyl human interleukin-2[alanine-125] (r-metHuIL-2[ala-125]) following intravenous and subcutaneous administration in rats.

Comparative pharmacotoxicity studies in rats were performed to evaluate the response to r-metHuIL-2[ala-125] following 2 or 4 weeks of daily intravenous or subcutaneous administration, as well as to evaluate pharmacokinetic and pharmacodynamic responses. Pharmacokinetic analysis indicated that r-metHuIL-2[ala-125] showed high bioavailability and nonlinear concentration profiles. Pharmacodynamic responses to intravenous or subcutaneous dosing with r-metHuIL-2[ala-125], as measured by white blood cell counts, were comparable. Preclinical safety studies (6, 30, and 150 micrograms kg-1 day-1) indicated that r-metHuIL-2[ala-125], whether given intravenously or subcutaneously, was associated with increased circulating and infiltrating levels of lymphocytes and eosinophils. Bone marrow lymphoid hyperplasia and splenic extramedullary hematopoiesis were similarly observed in each study. This pattern of effects was considered an exaggerated pharmacodynamic response to r-metHuIL-2[ala-125]. Of further note was a histopathologic finding described as hepatocyte single cell necrosis which was observed following both intravenous and subcutaneous administration and was considered to be a toxic response to high doses of r-metHuIL-2[ala-125]. The no observable adverse effect level (NOAEL) for r-metHuIL-2[ala-125] via intravenous administration was 6 micrograms kg-1 day-1, while that for subcutaneous administration was 30 micrograms kg-1 day-1. Data herein present a form of rHuIL-2 with pharmacokinetic and pharmacodynamic profiles that are similar when given by these two systemic routes. Pharmacotoxic data, based on NOAELs, suggest that subcutaneous administration may be a preferred clinical route of administration.

Absorption↗
PubMed1994

Characterization of disulfide linkages in platelet-derived growth factor AA.

Intermolecular and intramolecular disulfide linkages of recombinant human platelet-derived growth factor A chain dimer were determined by chemical methods including selective reduction-alkylation, peptide isolation, or detection of diphenylthiohydantoin derivative of cystine from Edman reactions. Cys-37 and Cys-46 were selectively reduced with reducing agents under native conditions and revealed to be involved in intermolecular bridges. Other disulfide linkages including Cys-10-Cys-54, Cys-43-Cys-91, and Cys-47-Cys-93 form intramolecular bridges. The disulfide structure is homologous to that of platelet-derived growth factor B chain dimer.

Alkylation↗
PubMed1994

Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds.

Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide-bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necessary for biological activity, since reduction of the dimer results in loss of activity and simultaneous conversion to monomeric form as determined by SDS-gel electrophoresis. However, reduction of the native molecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical for proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cysteines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Another mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in interchain disulfides were converted to serine, ran as a monomer on SDS-polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the activity of wild-type PDGF-BB disulfide-bonded dimer under physiological conditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weight analysis utilizing light scattering and sedimentation equilibrium demonstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conclude that proper formation of intrachain disulfide bonds is critical to maintaining the correct conformation of PDGF monomers, but that appropriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bonds thus appear to increase the stability of the PDGF dimer rather than being crucial to its existence.

Amino Acid Sequence↗
PubMed1994

Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor.

Keratinocyte growth factor (KGF), a recently discovered 18.9 kD member of the fibroblast growth factor family has been shown to selectively induce keratinocyte proliferation and differentiation in tissue culture. To explore its potential stimulating keratinocyte growth and differentiation in vivo, we analyzed for the influence of KGF on epithelial derived elements within a wound created through the cartilage on the rabbit ear. KGF accelerated reepithelialization (p = 0.004) and increased the thickness of the epithelium (p = 0.0005) when 4-40 micrograms/cm2 recombinant KGF was added at the time of wounding. The regenerating epidermis showed normal differentiation as detected by cytokeratin immunostaining. Remarkably, however, KGF stimulated proliferation and differentiation of early progenitor cells within hair follicles and sebaceous glands in the wound bed and adjacent dermis. There was a transient but highly significant increase in specific labeling of cycling cells in both basal and suprabasal layers that extended into the spinous layer of the regenerating epidermis. As an indication of specificity, the inflammatory cells and fibroblasts within the wound were not influenced by KGF. The results indicate that KGF is unique in its ability to accelerate reepithelialization and dermal regeneration by targeting multiple epithelial elements within the skin. These results suggest that KGF may induce specific epithelial progenitor cell lineages within the skin to proliferate and differentiate, and thus may be a critical determinant of regeneration of skin. Furthermore, these findings illustrate the potential capacity of this system to analyze epithelial differentiation programs and disorders of epidermis, dermal glandular elements, and hair follicles.

Animals↗
PubMed1994

Aggregation pathway of recombinant human keratinocyte growth factor and its stabilization.

Recombinant human keratinocyte growth factor (rhKGF) is prone to aggregation at elevated temperatures. Its aggregation pathway is proposed to proceed initially with a conformational change which perhaps results from repulsion between positively charged residues in clusters forming heparin binding sites. Unfolding of the protein leads to formation of large soluble aggregates. These soluble aggregates then form disulfide cross-linked precipitates. Finally these precipitates are converted to scrambled disulfides and/or non-disulfide cross-linked precipitates. Stabilizers such as heparin, sulfated polysaccharides, anionic polymers and citrate can greatly decrease the rate of aggregation of rhKGF at elevated temperatures. These molecules may all act by reducing charge repulsion on the protein thus stabilizing the native conformation. EDTA, on the other hand, is found to inhibit disulfide formation in aggregates and has only a moderate stabilizing effect on rhKGF.

Anions↗
PubMed1994

Direct assignment of disulfide bonds by Edman degradation of selected peptide fragments.

Disulfide linkages in peptides or proteins were analyzed by automated gas-phase Edman sequencing performed in minimized reducing agents. If cystine linkage was regulated at the same position in two peptides during peptide preparation, the diphenylthiohydantoin derivative of cystine was significantly recovered by Edman reaction. In contrast, when the crosslinked half cystines were present at different positions in the peptides, the derivative could be poorly detected. Upon direct sequence analysis of intact bovine insulin, the PTH derivatives of cystine from both Cys-A7 and Cys-B7 were significantly released after Edman cycle 7 and gave approximately 20% recovery of common PTH amino acids. However, Cys-A11 linked to Cys-A6 was poorly detectable after Edman cycle 11. For general use of this method, proteins need to be subjected to several sets of proteolytic or chemical cleavages in the hope that at least one of the fragments will have cystine linkage at the same position. This method was applied to several fragments of platelet-derived growth factor B chain and brain-derived neurotrophic factor.

Acetylation↗
PubMed1994

Structure and solubility of interleukin-2 in sodium dodecyl sulfate.

The solubility of an interleukin-2 (C125A) in an aqueous sodium dodecyl sulfate (SDS) solution was examined as a function of SDS concentration. A stock interleukin-2 solution at 1.1 or 0.31 mg/mL was mixed with one-tenth volume of a stock SDS solution at various concentrations. At intermediate SDS concentrations, the interleukin-2 showed nearly complete precipitation, while lower or higher SDS concentrations resulted in an increase in protein solubility. Circular dichroic analysis indicated that the protein remaining in the supernatant at low or high SDS concentrations has an altered conformation. Sedimentation velocity analysis of interleukin-2 in 0.1% SDS showed that about half the protein was monomeric, with the remainder as large aggregates with a broad size distribution from 20 to 100 S (M(r) approximately 10(5) - 10(7). In contrast, interleukin-2 in 0.01% SDS has a much smaller distribution of aggregates, sedimenting at ca. 2-10 S.

Chemical Phenomena↗
PubMed1994

The conformational stability of a non-covalent dimer of a platelet-derived growth factor-B mutant lacking the two cysteines involved in interchain disulfide bonds.

Native platelet-derived growth factor-B (PDGF-B) forms a covalent dimer through interchain disulfide bonds. In a previous study, an analog of PDGF-B was produced by replacing cysteine 43 and 52, which are involved in the interchain disulfide bonds, with serine. It was revealed that this analog protein has the dimeric molecule weight at pH 4 to 7, forming a non-covalent dimer in solution, and its mitogenic activity is similar to the native covalent dimer. However, the analog protein was more labile to pepsin digestion and low pH treatment, indicating that the interchain disulfides contribute to the stability of the protein. It is interesting to see if the conformation of the protein is affected by elimination of the interchain disulfide bonds, and if the interchain disulfides play any role in the stability of the protein. Circular dichroism and Fourier transform infrared spectroscopic analyses of the analog showed that it has a conformation similar to the wild type at pH 7.5, but is unfolded at pH 2.5, while the native PDGF-B disulfide-linked dimer shows an apparently unaltered conformation at pH 2.5. The analog is also less stable to sodium dodecylsulfate and guanidine HCl-induced denaturation at neutral pH. These results indicate that the non-covalent interactions are sufficient for proper folding and dimer formation at neutral pH, but that the interchain disulfide bonds greatly stabilize the native conformation of PDGF-B.

Circular Dichroism↗
PubMed1993

Disulfide bonds in recombinant human platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulfide linkages.

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.

Acetylation↗
PubMed1993

Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed.

Oh-eda et al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 [J. Biol. Chem. (1990) 265, 11,432-11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Conformation of glutathione adduct and oxidized forms of platelet-derived growth factor.

The conformational properties of several platelet-derived growth factors (PDGFs) were characterized by circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), gel filtration and sedimentation equilibrium. Three different forms of disulfide linked dimer, PDGF-AA, PDGF-AB, and PDGF-BB, showed similar far UV CD spectra with evidence for slight beta-structure, but little evidence of other regular secondary structures. These spectra were, however, different from the far UV CD spectra of the glutathione adducts of PDGF-A and B, suggesting that the latter two proteins adopt different conformations in the absence of intra- or inter-molecular disulfide bonds. FTIR studies confirmed this by showing that the glutathione adducts of the PDGF-B protein have a significantly lower amount of regular secondary structures than PDGF-BB. Additionally, the increased bandwidths of the amide I components of the FTIR spectrum of the glutathione adduct indicates a more flexible structure relative to the dimeric form. Sedimentation equilibrium analysis showed that PDGF-BB is primarily a dimer and that the glutathione form is primarily a monomer. Thus, it was concluded that the glutathione derivative has little affinity to form non-covalent dimers in neutral solution.

Chromatography, Gel↗
PubMed1992

High-performance anion-exchange chromatography of asparagine-linked oligosaccharides.

Oligosaccharides released enzymatically by N-glycanase from fetuin, alpha-acid glycoprotein, human chorionic gonadotropin, platelet-derived growth factor, and kallikrein were chromatographed on a polymeric pellicular anion-exchange column at pH values of 5 and 13. Separations occurred into groups of peaks containing the same number of sialic acids with an additional separation dependent upon the nature of the antennary structure present. High pH conditions were required for the optimum separation of fetuin oligosaccharides, while low pH conditions significantly improved resolution of oligosaccharides obtained from the other glycoproteins. The analytical separation of oligosaccharides under conditions of low pH has important implications in the development of chromatographic mapping and identification techniques for N-linked oligosaccharides present on recombinant proteins.

Animals↗